Director's Report to the National Advisory Council on Drug Abuse
May, 1999

Research Findings

Intramural Research

Molecular Neuropsychiatry Section, Cellular Neurobiology Branch

Methamphetamine-induced Increase of an Immediate Early Gene, c-fos, is Attenuated by Endogenous Opioid Peptide

Methamphetamine is a well-known psychostimulant of abuse that at high doses causes long-lasting neurotoxic effects. The mechanism underlying the neurotoxic action of methamphetamine is believed to involve free radical formation as well as the activation of certain immediate early genes. The immediate early genes are believed to be involved in the long-term effects of drugs of abuse by affecting the expression of other proteins that might be related to an altered state of physiology. In the past, the endogenous delta opioid peptide DADLE has been found by NIDA IRP scientists to prolong survival of peripheral organs such as the hearts and the lungs. NIDA IRP investigators also found that DADLE can block the dopaminergic terminal damage caused by methamphetamine and that DADLE can act as a free radical scavenger in vitro. More recent work has now found that DADLE blocks the increase of an immediate early gene c-fos in the area of brain that is most susceptible to the neurotoxic insult of methamphetamine. This constitutes the first report that a genomic influence exerted by a psychostimulant can be counteracted by an endogenous system. These data will be useful for understanding more about the physiological role of endogenous opioid peptides in the brain and pave an avenue for developing certain therapeutic approach for treating methamphetamine abuse. Hayashi, T., Tsao, L.-I., Cadet, J.L. and Su, T.-P. European Journal of Pharmacology, 366, pp. R7-R8, 1999.

Brain Imaging Section, Neuroimaging Branch

Age-dependent Dopamine Receptor Loss in Humans may be Overestimated

The relation between striatal dopamine D2 receptor binding and aging was investigated in rhesus monkeys with positron emission tomography (PET). Monkeys, ranging in age from 39 to 360 months, were scanned with 11C-raclopride and binding potential in the striatum was estimated graphically. Because magnetic resonance imaging analysis revealed a concomitant relation between size of striatum and age, the dynamic PET data were corrected for possible partial volume artifacts. The age-related decline in binding potential was 1% per year and, importantly, was smaller than the apparent effect if the age-related change in size was ignored. The rate of decline in binding potential is consistent with in vitro findings in monkeys but smaller than what has been measured previously in humans using PET. Previous PET studies in humans, however, have not corrected for partial volume error, although a decline in striatal size with age has been demonstrated. The results of this study suggest that partial volume correction must be applied to PET data to accurately detect small changes in receptor binding that may occur in parallel with structural changes in the brain in order not to overestimate the magnitude of the dopamine receptor loss. Morris, E.D., Chefer, S.I., Lane, M.A., Muzic Jr., R.F., Wong, D.F., Dannals, R.F., Matochik, J.A., Bonab, A.A., Villemagne, V.L., Grant, S.J., Ingram, D.K., Roth, G.S., and London, E.D. Loss of D2 Receptor Binding with Age in Rhesus Monkeys: Importance of Correction for Differences in Striatal Size. J. Cereb. Blood Flow & Metab., 19(2), pp. 218-229, 1999.

Synthetic Route Developed for a Radiotracer Suitable for Imaging Nicotinic Acetylcholine Receptors in the Brain using SPECT

Interest in the normal and pathological roles of nicotinic acetylcholine receptors (nAChRs) within the brain has created a need for selective radiolabeled ligands that can be used to noninvasively image these receptors. This need has been addressed by devising simple radiochemical syntheses for 5-[125I]iodo-3-(2(S)-azetidinylmethoxy)pyridine (5-[125I]iodo-A-85380) as well as 5-[123I]iodo-A-85380, by radioiodination of 5-trimethyltin-3-((1-tert-butoxycarbonyl-2(S)-azetidinyl)methoxy)pyridine, followed by acidic deprotection. The average radiochemical yield was 50-65% and the average specific radioactivities of the I-125 and I-123 containing ligands were 2000 mCi/Ámol and 7000 mCi/Ámol, respectively. The binding affinity of both iodinated radioligands for nicotinic receptors was exceptionally high, 11.2 pM. Preliminary in vivo studies and ex vivo autoradiography in mouse brain demonstrated that 5-[125I]iodo-A-85380 selectively labels nicotinic receptor-rich regions with very high potency and specificity. These studies demonstrate the suitability of 5-[123I]iodo-A-85380 as a radioligand for single photon emission computed tomography (SPECT) imaging of nicotinic receptors within the brain. Horti, A.G., Koren, A.O., Lee, K.S., Mukhin, A.G., Vaupel, D.B., Kimes, A.S., Stratton, M., and London, E.D. Radiosynthesis and Preliminary Evaluation of 5-[123/125I]Iodo-3-(2(S)-azetidinylmethoxy)-pyridine: A Radioligand for Nicotinic Acetylcholine Receptors. Nucl. Med & Biology, 26, pp. 175-182, 1999.

Chemistry & Drug Metabolism Section, Clinical Pharmacology Branch

Intensity-Related Effects of Imagery Scripts on Tobacco Craving and Mood

Two experiments were conducted to determine whether active imagery would elicit tobacco craving in smokers with histories of drug abuse who were not interested in quitting smoking. In Experiment 1, we used scripts that contained positive, negative, or neutral affective content with and without descriptions of smoking urge. Scripts with urge content and negative affect scripts increased subjective reports of tobacco craving. An interaction between affective manipulation and urge content was observed on self-reported mood. In Experiment 2, positive affect scripts that varied in amount of urge content produced an orderly increase in tobacco craving as a function of urge intensity, suggesting that changes were specific to the imagery manipulation. In both experiments, increases in tobacco craving were positively correlated with craving for drug of choice, suggesting that stimuli that engender smoking urges may occasion craving for other drugs of abuse. Harris, N.A., Taylor, R.C., Singleton, E.G., and Heishman, S.J., Poster, Society for Research on Nicotine and Tobacco, San Diego, California, 6 March 1999.

Behavioral Pharmacology Section, Preclinical Pharmacology Laboratory

The prevalence of tobacco smoking and coffee drinking place nicotine and caffeine among the most used licit drugs in many societies and their consumption is often characterized by concurrent use. The pharmacological basis for any putative interaction between these drugs remains unclear. Epidemiological reports support anecdotal evidence, which suggests that smokers consume caffeine to enhance the euphoric effects of nicotine. The scientists have recently examined the effects of chronic exposure to caffeine on self-administration responding maintained by nicotine in Sprague-Dawley rats. Rats consuming caffeine (approximately 150-180 mg/kg per day) in their drinking water for 7 days prior to the beginning and throughout behavioral testing acquired intravenous nicotine self-administration (0.03 mg/kg per infusion) more rapidly than did controls. In a cross-over design, exclusion of caffeine brought levels of nicotine self-administration back to baseline, while adding caffeine to the drinking water of control rats increased responding maintained by nicotine over 90%. These findings strongly suggest that caffeine can potentiate the reinforcing properties of nicotine, thus highlighting the importance of environmental factors in shaping and maintaining tobacco smoking. Shoaib, M., Swanner, L.S., Yasar, S. and Goldberg, S.R. Psychopharmacology, 142, pp. 327-333, 1999.

Behavioral Effects of Nicotine, Amphetamine and Cocaine Under a Fixed-interval Schedule of Food Reinforcement in Rats Chronically Exposed to Caffeine

Epidemiological surveys demonstrate that caffeine, the main psychoactive ingredient of coffee, is a positive correlate in drug abuse. To characterize the behavioral nature of caffeine interactions with other psychomotor stimulants, IRP scientists examined the effects of chronic caffeine exposure on the behavioral responses to nicotine, amphetamine, cocaine, the selective D1 agonist SKF-82958 and the selective D2 receptor agonist NPA, in rats responding under a fixed interval (FI) schedule of food reinforcement. Following stabilization of responding, Sprague-Dawley rats were divided into two groups; one maintained on tap water (control) and the other on caffeine (3 mg/ml added to the drinking water). Rats developed complete tolerance to the effects of caffeine in the drinking water over 5 days of caffeine exposure. After behavior stabilized, effects of drugs were evaluated. Nicotine, amphetamine and cocaine each produced biphasic dose-dependent changes in response rate with maximum increases in response rate following immediate doses and decreases in response rates following higher doses. The increase in rates of responding produced by amphetamine or cocaine (but not nicotine) were greater in caffeine-drinking than in water-drinking rats. Both SKF-82958 and NPA produced only dose-dependent decreases in rates of responding. Caffeine-drinking rats were less sensitive to the rate-depressant effects of SKF-82958 than water-drinking rats. However, similar changes were produced by NPA in both groups. Chronic exposure to caffeine produced complete insurmountable tolerance to the response-rate increasing (stimulant) effects of acute caffeine in caffeine-drinking rats. In conclusion, this study revealed that chronic caffeine exposure potentiates the behavioral response to amphetamine and cocaine but not to that of nicotine in rats responding under a FI schedule of food reinforcement. Thus, it is likely that these effects are mediated through different pharmacological mechanisms. Jaszyna, M., Gasior, M., Shoaib, M., Yasar S. and Goldberg, S.R. Psychopharmacology, 140, pp. 257-271, 1998.

Alteration of the Discriminative Stimulus Effects of Nicotine in Rats by Chronic Exposure to Caffeine

Caffeine and nicotine are the main psychoactive ingredients of coffee and tobacco, with a high frequency of concurrent use in humans. IRP scientists examined the effects of chronic caffeine exposure on the acquisition and the pharmacological characteristics of a nicotine discrimination in Sprague-Dawley rats. Once rats learned to lever-press reliably under a fixed-ratio schedule for food pellets, they were divided into two groups; 12 animals were maintained continuously on caffeine (3 mg/ml) added to the drinking water and another 12 control rats continued to drink tap water. Both water- and caffeine-drinking groups required a comparable number of training sessions to attain reliable stimulus control. The nicotinic-receptor antagonist mecamylamine blocked the discriminative effects of nicotine with comparable potency and efficacy in water- and caffeine-drinking groups. There was a dose-related generalization to nicotine cue in water-drinking rats after i.p. treatment with d-amphetamine, cocaine, the selective dopamine uptake inhibitor GBR-12909, apomorphine, and the selective dopamine D1 receptor agonist SKF-82958, but not in caffeine-drinking rats. There was no generalization to the nicotine cue after i.p. treatment with caffeine or the selective D2 (NPA) and D3 (PD 128,907) dopamine-receptor agonists in either water- or caffeine-drinking rats. The dopamine-release inhibitor CGS 10746B reduced the discriminative effects of nicotine in water-drinking rats, but not in caffeine-drinking rats. Thus, chronic caffeine exposure (average, 135 mg/kg/day) did not affect the rate of acquisition of the nicotine discrimination, but it did reduce the dopaminergic component of the nicotine-discriminative cue. The reduction was permanent, as this effect was still evident after the caffeine solution was replaced with water in caffeine-drinking rats. That nicotine could reliably serve as a discriminative stimulus in the absence of the dopaminergic component of its discriminative cue may differentiate nicotine from "classical dopaminergic" drugs of abuse such as cocaine and amphetamine. Gasior, M., Shoaib, M., Yasar, S., Jaszyna, M. and Goldberg, S.R. Journal of Pharmacology and Experimental Therapeutics, 288, pp. 1053-1073, 1999.

Effects of Dopamine- and Serotonin-releasing Agents on Methamphetamine Discrimination and Self-administration

Although most behavioral effects of methamphetamine are mediated by the dopaminergic neurotransmitter system, neurochemical findings suggest that there also is strong involvement of serotonergic neurotransmission. To analyze the relative involvement of dopamine and serotonin in the behavioral properties of methamphetamine, IRP scientists tested two amphetamine analogs that selectively release either brain dopamine (phentermine) or serotonin (fenfluramine) in Sprague Dawley rats. These compounds were tested for their ability to substitute for methamphetamine in rats trained to discriminate methamphetamine from saline and, subsequently, for their ability to alter methamphetamine self-administration. The dopamine releaser phentermine decreased methamphetamine self-administration, but only at a dose that fully generalized to the methamphetamine stimulus in the discrimination study. The serotonin releaser fenfluramine attenuated methamphetamine self-administration to a much larger extent than phentermine and did so at doses that did not generalize to methamphetamine and did not decrease rate of responding for food. These results suggest that dopamine release plays a dominant role in the subjective effects of methamphetamine. However, stimulation of serotonin release can strongly modify methamphetamine self-administration. Munzar, P., Baumann, M.H., Shoaib, M. and Goldberg, S.R. Psychopharmacology, 141, pp. 287-296, 1999.

Potentiation of the Discriminative-stimulus Effects of Methamphetamine by a Histamine h4 Receptor Antagonist

Most behavioral effects of methamphetamine appear to be related to the release of dopamine. Recent findings, however, suggest that histamine release may also contribute to its actions. Endogenous histamine release is regulated by histamine h4 autoreceptors. Their blockade by the h4 antagonist thioperamide may increase histamine release, thus augmenting methamphetamine's behavioral effects. To test this hypothesis, IRP researchers tested thioperamide in rats discriminating methamphetamine from saline. Thioperamide potentiated the discriminative-stimulus effects of methamphetamine and this effect was reversed by co-administration of R-alpha-methylhistamine, a h4 agonist. These data suggest that methamphetamine's behavioral effects are modified by drugs affecting histamine release. This knowledge may result in the development of new strategies for the treatment of psychostimulant abuse. Munzar, P., Nosal, R. and Goldberg, S.R. European Journal of Pharmacology, 363, pp. 93-101, 1998.
Discrimination and Self-administration of Nicotine by Inbred Strains of Mice The discriminative stimulus effects of nicotine have been characterized by IRP scientists in two inbred strains of mice that differ in many pharmacological responses. They have also investigated the feasibility of intravenous self-administration studies with nicotine in one of the strains. For discriminative studies, three groups of C57BL/6 and one group of DBA/2 mice were trained in a two-lever operant conditioning paradigm with a tandem schedule of food reinforcement. After 40 sessions, accuracy reached 57.5, 77.5 and 90.0% in C57BL/6 mice trained with (-)-nicotine (SC) in doses of 0.4, 0.8 and 1.6 mg/kg, respectively. DBA/2 mice trained with 0.8 mg/kg nicotine attained similar (73.3%) accuracy. All groups of mice yielded orderly dose-response curves for nicotine. Rates of responding in the undrugged state were lower for DBA/2 than for C57BL/6 mice; DBA/2 mice were also slightly less sensitive than C57BL/6 mice to the response rate-reducing effect of nicotine. The nicotine antagonist mecamylamine blocked the discriminative stimulus effect of nicotine in all groups. In the intravenous self-administration study, nicotine served as a positive reinforcer in drug-naive C57BL/6J mice. The methods described provide possible approaches for genetic analyses of strain differences in sensitivity to the discriminative and reinforcing stimulus properties of nicotine. Stolerman, I.P., Naylor, C., Elmer, G.I. and Goldberg, S.R. Psychopharmacology, 141, pp. 297-306, 1999.

Clinical Trials Section, Treatment Branch

Occurrence of Cocaine in Urine of Substance Abuse Treatment Patients

As part of ongoing research efforts to improve methods of monitoring drug use in treatment patients, the presence of cocaine in urine specimens was evaluated as a possible marker for recent illicit cocaine use. A total of 2,327 urine specimens collected during a clinical trial of a cocaine abuse treatment study were tested. Cocaine was measured by gas chromatography-mass spectrometry and benzoylecgonine equivalents were determined by fluorescence polarization immunoassay (FPIA). More than one-third of the specimens were positive (>25 ng/mL) for cocaine, and nearly two-thirds were positive (> 300 ng/mL) for cocaine metabolite. Median concentrations of cocaine and benzoylecgonine equivalents were 235 and 14,900 ng/mL and maximum concentrations were 112,025 and 1,101,190 ng/mL in cocaine- or benzoylecgonine-positive specimens, respectively. No significant differences in cocaine or benzoylecgonine concentrations between White and African American or between male and female patients were found. Cocaine was present less frequently and at lower concentrations than benzoylecgonine, but more frequently than expected based on an approximate average half-life of 1 hour. These results suggest that cocaine may exhibit a longer terminal half-life and/or that accumulation of cocaine can occur in chronic, heavy users. Preston, K.L., Goldberger, B.A., and Cone, E.J. Occurrence of Cocaine in Urine of Substance Abuse Treatment Patients. Journal of Analytical Toxicology, 22, pp. 580-586, 1998.

The Influence of an Instruction on the Stimulus Effects of Drugs in Humans

Volunteers (N = 14) participated in two experiments differing only in instructional set using a standard drug discrimination paradigm. In each experiment subjects were trained to discriminate between 75 mg tripelennamine and placebo in 15 training sessions. In one experiment (SED), participants were instructed that one of the two drugs was more sedative-like than the other drug and during the other experiment (STIM), one of the two drugs was more stimulant-like. The experiments were otherwise the same. Order of presentation of the experiments was counterbalanced across participants. During generalization tests, participants received capsules that contained diazepam (2.5 and 5 mg) or d-amphetamine (5 and 10 mg). Tripelennamine and placebo were discriminated on approximately 65% of sessions with no differences across experiments. Tripelennamine increased in scores on subjective effects scales measuring sedative effects and decreased in scores on scales measuring stimulant effects across both experiments. Amphetamine and diazepam each produced typical changes in stimulant and sedative effects, respectively, with only minor differences between experiments. Some subjective effects of the training and test drugs differed between experiments with more sedative and less stimulant and anxiety effects during SED relative to during STIM. In SED and STIM, capsules were labeled over 80% of the time as a sedative and stimulant, respectively. These results indicate that instructions designed to give participants expectations as to the types of drugs they would be administered produced no effect on discrimination and only a few significant changes in subjective effects. On the other hand, when participants were asked to name (label) the drug they believed they received, their answers reflected the instructional set. Johanson, C.-E., and Preston, K.L. The Influence of an Instruction on the Stimulus Effects of Drugs in Humans. Experimental and Clinical Psychopharmacology, 6, pp. 427-432, 1998.

Clinical experience has shown that poor compliance with naltrexone treatment seriously compromises its clinical utility. This study tested the efficacy of a voucher-based incentive program for improving compliance with naltrexone treatment. Opioid-dependent patients who had recently completed opioid withdrawal treatment were enrolled in a 12-week outpatient treatment research program in which they received naltrexone three times weekly and counseling once weekly. Subjects were randomized to three treatment groups: 1) Contingent (N = 19) - vouchers earned for each ingested naltrexone dose; 2) Non-Contingent (N = 19) - vouchers given at rates and values matched to the Contingent Group but independent of naltrexone ingestion; and 3) No Vouchers (N = 20) - no vouchers were available. The vouchers had monetary value and were exchangeable for goods and services. The Contingent group had significantly longer treatment retention and ingested significantly more doses of naltrexone (consecutive and total) than either control group. Voucher incentives can significantly increase adherence to naltrexone maintenance in recently detoxified opioid dependent individuals. Preston, K.L., Silverman, K., Umbricht, A., DeJesus, A., Montoya, I.D., and Schuster, C.R. Improvement in Naltrexone Treatment Compliance with Contingency Management. Drug and Alcohol Dependence, 54, pp. 127-135, 1999.

Cellular Neurophysiology Section, Cellular Neurobiology Branch

Phenotypic Characterization of GFR alpha-1 Expressing Neurons

The glial cell line-derived neurotrophic factor (GDNF) rescues motoneurons and nigral dopaminergic (DA) cells from induced cell death in different species of animals. In addition, GDNF results in a remarkable improvement of behavioral functions in Parkinsonian monkeys. A high affinity receptor for GDNF has been cloned (GFR alpha-1), whose pattern of expression indicates that it is not restricted to those brain areas containing motoneurons and DAergic cells. These observations suggest that GDNF may activate a number of different sub-types of brain cells. IRP scientists have found that GFR alpha-1 is expressed in defined populations of neurons. As anticipated, GFR alpha-1 occurs in DAergic cells of the substantia nigra compacta (SNC) and the ventral tegmental area (VTA). GFR alpha-1 is also present in GABAergic cells located either in the lateral SNC or in the substantia nigra reticulata (SNR), and these GABAergic cells are more abundant in the caudal levels of the SNR. Results imply that GDNF-induced improvements in the behavior of semi-parkinsonian animal models may, in part, involve GABAergic cells of the SNR. Using either a cerebral ischemia model (middle cerebral artery, MCA ligation) or physical cortical injury, IRP investigators found a robust up-regulation of GFR alpha-1 expression in granule cells ipsilateral to the area receiving the insult. In both models, GFR alpha-1 up-regulation peaks 6 hours following ischemia or physical injury. These results indicate that GFR alpha-1 might be part of an endogenous neuroprotective system that is activated following brain insult or injury and may act as a defense mechanism in the brain. Okada, Y. et al. Experimental Implication of Celiac Ganglionotropic Invasion of Pancreatic-Cancer Cells Bearing C-Ret Proto-Oncogene with Reference to Glial-Cell-Line-Derived Neurotrophic Factor (GDNF). Int J Cancer, 81(1), pp. 67-73, Mar 31, 1999. Perez-Navarro, E. et al. Intrastriatal Grafting of a GDNF-Producing Cell Line Protects Striatonigral Neurons from Quinolinic Acid Excitotoxicity In Vivo. Eur J Neurosci. 11(1), pp. 241-249, Jan 1999. Enokido, Y. et al. GFR Alpha-4 and the Tyrosine Kinase Ret Form a Functional Receptor Complex for Persephin. Curr Biol. 8(18),
pp. 1019-1022, Sep 10, 1998. Trupp, M. et al. Multiple GPI-Anchored Receptors Control GDNF-Dependent and Independent Activation of the C-Ret Receptor Tyrosine Kinase. Mol Cell Neurosci. 11(1-2), pp. 47-63, May 1998. Hishiki, T. et al. Glial Cell Line-Derived Neurotrophic Factor/Neurturin-Induced Differentiation and its Enhancement by Retinoic Acid in Primary Human Neuroblastomas Expressing C-Ret, GFR Alpha-1, and GFR Alpha-2. Cancer Res. 58(10), pp. 2158-2165, May 15, 1998.